Myopia has been rapidly increasing in recent years worldwide [1
]. Holden et al. suggested that myopia as well as high myopia would increase in the next 30 years estimating that the prevalence of myopia and high myopia would reach 5 billion and 1 billion, respectively, and that 40% of blindness would be attributed to myopia [1
]. As the number of myopia increases, the number of high myopia and pathological myopia which may lead to myopic maculopathy, retinal detachment, and glaucoma is expected to increase [1
]. The medical cost related to the increase of myopia will expand, and therefore, suppression of myopia is now more important [2
]. Development of the way of myopia prevention is much more aggressively performed than before [3
]. Although preventative measures such as outdoor activities, atropine eye drops, and orthokeratology lenses are under investigation [5
], there have few oral medicines or supplements which showed significant suppressive effect on myopia progression.
The light environment was considered to be a significant factor for myopia progression [6
]. Violet light is 360 to 400 nm wavelength in the visible light and shown to have a suppressive effect of myopia [9
]. Furthermore, we reported that crocetin suppressed myopia progression in mice through the same mechanism as violet light exposure [12
Crocetin was demonstrated to have a suppressive effect on myopia progression in mice, and the expression of Egr-
1, a myopia suppressive gene, was shown to be in a dose-dependent manner in vitro [12
]. In the previous study, crocetin-containing extract chow with two different concentrations, 0.003% and 0.03%, showed an equally suppressive effect in a murine model of lens-induced myopia [12
]. Regarding lens-induced myopia, when equipped with minus diopter lenses, the axial length elongates to adjust the focus of the vision on the retina, which consequently makes the eye myopic. We previously revealed and reported that −30 D lenses are the most effective to produce lens-induced myopia phenotypes in mice after examining several lenses with different diopter, from −30 D to +5 D, to find which diopter is the most effective [13
]. As it meant 0.003% of crocetin-containing extract was sufficient, the minimum required dose of crocetin to suppress myopia progression should be investigated for applying crocetin administration to human children and performing a randomized controlled trial. In the current study, we evaluated the animal model fed with either normal or two lower concentrations (0.001% and 0.0003%) of crocetin-containing extract chow.
Previous studies demonstrated that both of the concentrations, 0.003% and 0.03%, of crocetin-containing extract equally have a suppressive effect on myopia progression [12
]. Due to this result, we investigated the minimum required dose to suppress myopia progression in mice. As a result, 0.0003% of crocetin-containing extract showed a suppressive effect on myopic refractive change but it showed no suppressive effect on axial elongation, whereas 0.001% of crocetin-containing extract showed an effect on both refractive change and axial elongation. This result gave us the idea that the critical point existed between these concentrations.
The concentration of crocetin detected in the eyeballs with 0.0003% crocetin-containing extract chow was not different from that with control chow. Meanwhile, when 0.001% crocetin-containing chow was administered, the concentration in the eye balls was significantly different from those with 0.003% crocetin-containing extract chow as well as the control chow, which corresponded to phenotypes of refraction and AL and supported the myopia suppressive effect of 0.001% crocetin-containing extract chow.
We prepared the concentrations of crocetin-containing chows so that 3 mg/kg/day of Crovit P were administered when a three-week-old B6 mouse consumes 0.001% of crocetin-containing extract per day. Crovit P used in this study is a product which guarantees more than 75% purity of crocetin. In the meantime, the body surface area of a 10 g body weight mouse is 0.0035 m2, whereas the body surface area of a 10 year-old child, who is thought to be predisposed to myopia progression, is generally 1.15 m2. We estimated the body surface area of an average 10 year-old child using the Du Bois and Du Bois formula based on the statistical survey of school health in 2018 in Japan. Consequently, 0.0001% crocetin-containing extract chow corresponds to 10 mg/day of Crovit P, which exceeds 7.5 mg/day of pure crocetin.
When excerpted from previous human studies on crocetin administration, the dose varies between 7.5 mg to 22.5 mg [14
]. 0.0001% crocetin-containing extract chow, the minimum required dose to suppress myopia, corresponds to 7.5 mg/day when administered to human. Umigai et al. reported that 7.5 mg to 22.5 mg of crocetin can be safely administered to human without any side effects [14
The question whether the age of mice and the period of administration of crocetin are proper can be raised. Regarding the age, mice at three weeks of age, which corresponds to the adolescent period in humans, were most susceptible to experimental myopia induction [13
]. In addition, the period of three weeks to induce myopia by applying −30 diopter lenses in mice is found to be good enough to obtain statistical significant differences [13
]. As for the timing of the initiation of crocetin administration to human children, it is presumed that the treatment should be started by their adolescence.
The difference in AL between the control group and 0.001% crocetin-containing chow group was 0.06 mm with a statistical significance. The mean murine ocular axial length is approximately 3.264 ± 0.047 mm measured by a SD-OCT [15
]. The human full-term newborn eye has a mean axial length of 16–18 mm [16
]. The mean human adult values for axial length are 22–25 mm and mean refractive power −25.0 ± 1.0 D [16
]. The difference in AL of 0.06 mm in mice corresponds to approximately 0.43 mm in humans, which can be considered clinically significant.
In this study, crocetin was detected in the eyes of mice fed with crocetin in a dose-dependent manner, whereas a small amount of crocetin was also found in the eyes of control mice fed with normal chows which contained no crocetin. To exclude the possibility of contamination, crocetin in the eyes of mice bred in other facility which is free from crocetin was measured; consequently, crocetin was also detected in the eyes of these mice. The crocetin detected in the eyes of control mice were thought to be derived from the pigments which naturally exists in the retina. Bowmaker et al. previously reported that pigments which was considered to be crocetin existed in the retina [17
]. There have been few reports regarding pharmacodynamics of crocetin in the eyes, and the efficacy and the role of crocetin have not sufficiently been proven.
This study has some limitations. Though extracts of crocetin are mixed to chows in this research, it is technically difficult to prepare low concentrations, less than 0.0003%, of crocetin-containing extract chow. This should be further investigated.
This research was fundamentally aimed at application of administration of crocetin to humans. Myopia has been increasing worldwide and is anticipated to continue to increase. Possible preventative measures against myopia progression and treatments for myopia are now eagerly under investigation. Crocetin can be one of the hopeful candidates as a myopia preventive method and expected to be applied to humans. We hope our study will help future investigation of myopia prevention.
Patents have been registered for the therapeutic effects of crocetin (patent no. 6,502,603 by Tsubota Laboratory, Inc., Tokyo, Japan, and Rohto Pharmaceutical Co., Ltd., Osaka, Japan.) and applied for the design of the mouse eyeglass by Tsubota Laboratory, Inc. (patent application no. 2017-41349).